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WO2009147246A1 - Composés permettant de réduire ou d'inhiber l'expression de pkd1 pour le diagnostic et le traitement des tumeurs du cerveau - Google Patents

Composés permettant de réduire ou d'inhiber l'expression de pkd1 pour le diagnostic et le traitement des tumeurs du cerveau Download PDF

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Publication number
WO2009147246A1
WO2009147246A1 PCT/EP2009/056972 EP2009056972W WO2009147246A1 WO 2009147246 A1 WO2009147246 A1 WO 2009147246A1 EP 2009056972 W EP2009056972 W EP 2009056972W WO 2009147246 A1 WO2009147246 A1 WO 2009147246A1
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Prior art keywords
seq
expression
cells
homologue
isolated polynucleotide
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PCT/EP2009/056972
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English (en)
Inventor
Eva Bernhart
Hans Eder
Manuel Mrfka
Wolfgang Sattler
Andreas Zimmer
Daniela Reischl
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Medizinische Universität Graz
Universität Graz
Jsw Life Sciences Gmbh
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Application filed by Medizinische Universität Graz, Universität Graz, Jsw Life Sciences Gmbh filed Critical Medizinische Universität Graz
Priority to EP09757627A priority Critical patent/EP2297311A1/fr
Publication of WO2009147246A1 publication Critical patent/WO2009147246A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11013—Protein kinase C (2.7.11.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

  • the present invention relates inter alia to a role of PKDl in the progression of malignancy in brain tumors.
  • the invention provides compounds suitable for reducing or inhibiting the expression of PKDl.
  • the invention further relates to the use of said compounds for the diagnosis and therapy of cancer, in particular brain cancer.
  • Gliomas a type of primary central nervous system tumors arising from glial cells, are the most common primary brain tumors in humans and occur at an incidence of almost 12 per 100,000 people with a male predominance.
  • Astrocytomas which are derived from astrocytes, are the most common type of gliomas.
  • the World Health Organization has graded gliomas into 4 grades according to histopathology.
  • Malignant gliomas refer to WHO grade III (anaplastic astrocytoma) and IV (glioblastoma multiforme).
  • WHO grade III anaplastic astrocytoma
  • IV glioblastoma multiforme
  • DA et al. (2006), J. Clin. Oncol. 24, 1253-1265 These high grade gliomas carry a poor prognosis, causing death within a few weeks if left untreated. Symptoms depend on which part of the brain is affected by the tumor and include headaches, nausea, vomiting, disturbed vision, convulsions and muscle spasms.
  • the current standard of care for malignant gliomas consists of surgery, radiotherapy and conventional chemotherapies.
  • the oral alkylating agent temozolomide has become a standard chemotherapy drug for the treatment of malignant gliomas.
  • PKC ⁇ Protein kinase D 1
  • PRKDl Protein kinase D 1
  • PKC ⁇ Protein kinase D 1
  • RNAi mediated knockdown of Protein kinase Dl leads to growth inhibition of brain tumors, in particular of astrocytoma grade III and IV.
  • the present invention thus relates inter alia to a role of PKDl (also known as PRKDl) in the progression of malignancy in brain tumors and to the use of an anti- PKDl therapeutic approach to combat tumors in general, and malignant gliomas in particular.
  • PKDl also known as PRKDl
  • the present therapeutic approach is in particular based on the use of anti-PKDl tools relating to RNA interference- (RNAi), antisense-, expression vector- , or any other related approaches aiming to reduce or inhibit the expression or activity of PKDl in tumor cells, in particular in malignant glioma cells, including reducing or inhibiting the expression or activity of PKDl by means of aptameres, specific inhibitory peptides or specific antibodies directed against PKDl.
  • RNAi RNA interference-
  • antisense- antisense-
  • expression vector- or any other related approaches aiming to reduce or inhibit the expression or activity of PKDl in tumor cells, in particular in malignant glioma cells, including reducing or inhibiting the expression or activity of PKDl by means of aptameres, specific inhibitory peptides or specific antibodies directed against PKDl.
  • the present invention relates to a compound suitable for reducing or inhibiting the expression or activity of a polypeptide according to SEQ ID NO: 3 or a homologue thereof.
  • the present invention provides an isolated polynucleotide, comprising (a) a first polynucleotide sequence comprising SEQ ID NO: 4 or a fragment or derivative thereof and a second polynucleotide sequence comprising SEQ ID NO: 1 or a fragment or derivative thereof; or
  • SEQ ID NO: 2 or a fragment or derivative thereof.
  • the invention further provides an expression vector comprising an isolated polynucleotide according to the invention.
  • the present invention also relates to a host cell comprising an expression vector according to the invention.
  • the present invention relates to a carrier particle comprising at least one compound according to the invention that is suitable for reducing or inhibiting the expression or activity of a polypeptide according to SEQ ID NO: 3 or a homologue thereof and/or an expression vector or host cell according to the invention.
  • the present invention relates to a pharmaceutical composition for the treatment of cancer comprising at least one compound according to the invention that is suitable for reducing or inhibiting the expression or activity of a polypeptide according to SEQ ID NO: 3 or a homologue thereof and/or an expression vector, host cell or carrier particle according to the invention.
  • the present invention in a further aspect also relates to a transgenic animal containing an expression vector capable of expressing a polypeptide which is identical to or a homologue of the polypeptide according to SEQ ID NO: 3 and wherein the polypeptide has the molecular function of the polypeptide according to SEQ ID NO: 3.
  • the present invention relates to a method of detecting the presence of a tumor in a biological sample from a subject comprising at least the steps of:
  • NO: 3 or the homologue thereof in comparison to the control indicates the presence of a tumor in the subject.
  • the present invention also relates to a method for identifying a molecule capable of modulating cell proliferation comprising at least the following steps:
  • FIGURE LEGENDS determining the activity or the expression level of a polypeptide according to SEQ ID NO: 3 or a homologue thereof in said cells, (d) comparing the activity or the expression level in (c) to the activity or expression level in a control, wherein a higher or lower activity or expression level of the polypeptide according to SEQ ID NO: 3 in comparison to the control indicates that the test compound is capable of modulating cell proliferation.
  • Fig.l PKDl protein content correlates with tumor grading.
  • Proteins from A 172 cells and tumor specimens of the indicated WHO grading were extracted, separated by SDS-PAGE, electrob lotted and detected using a anti PKDl- antiserum. ECL was used to visualize immunoreactive bands. The number of samples is indicated, results are expressed as % of band intensity observed in Al 72 cells.
  • Fig. 2 Immunohistochemial characterization of PKDl expression in a glioblastoma sample.
  • Cryosections of the tumor sample were thawed, fixed in acetone and rehydrated in PBS. After blocking, the sections were incubated with rabbit anti-PKDl, HLA-DR, and GFAP IgG, followed by the corresponding secondary antibodies. Samples were mounted with Moviol and analyzed on a confocal laser-scanning microscope. The x/y dimension of the scanned field is 47.23 ⁇ m.
  • Fig. 3 PMA-induced translocation of PKDl-GFP.
  • a PKDl-GFP construct was transiently over-expressed in A 172 cells.
  • the images show cells before (A) and after incubation with PMA (100 nM) for up to 30 min (B- H, 5 min intervals).
  • PMA 100 nM
  • PKDl trafficking is observed to the plasma membrane where it is mainly located in cellular pseudopodia/invadopodia
  • Fig. 4 PDGF-induced translocation of PKDl.
  • a PKDl-GFP construct was transiently overexpressed in Al 72 cells. Cells were stimulated with PDGF (30 ng/ml) and cells were visualized after 2(A), 5 (B), 10 (C), 15 (D), and 20 (E) min. Arrows indicate sites of PKDl accumulation in response to PDGF.
  • Lysophosphatidic acid affects A 172 cell growth.
  • Fig. 6 LPA-dependent activation of PKDl in A 172 and primary glioblastoma (GBM) cells.
  • Fig. 7 Silencing of PKDl in Al 72 cells
  • Fig. 9 A 172 glioblastoma cells express all members of the PKD family.
  • PKDl F: ATT CCT TCT CTC CTC CTC CT, (SEQ ID NO: 8), R: ATA CTG AGG
  • PKD2 F: ATC CTC TTC CTC CCT CTT CTG (SEQ ID NO: 10), R: CAC AAG
  • PKD3 F: AGT CAC ATG TCC ACC AGG AA (SEQ ID NO: 12), R: TGA GGA GTA ACA GGC ATG AG (895 bp); (SEQ ID NO: 13)
  • PCR products were detected at the expected sizes (582, 667, and 895 bp, respectively). Identity was confirmed by sequencing.
  • Fig. 10 Silencing of PKDl in primary glioblastoma cells Primary cells were electroporated with siRNA (3 ⁇ g) from Biospring. At the indicated time points cells were lysed and cellular PKDl levels were assessed by Western blotting in duplicates
  • Fig. 11 Panther classification of induced and repressed genes in PKDl silenced cells
  • the genes were grouped on the basis of the PANTHER classification system according to their involvement in particular biological processes. It is of importance that in the list of upregulated genes p21 was found that could be responsible for repressed growth in silenced A 172 cells. In the list of downregulated genes it is obvious that some representatives that contribute to proteolysis (a process thought to favor invasiveness of tumor cells) was downregulated in PKDl silenced cells.
  • Fig. 12 2D-DIGE analysis of untreated and scrambled or PKDl-siRNA transfected Al 72 cells.
  • Fig. 14 pH-dependent size of ternary nanoparticles
  • Fig. 15 Effects of desalted siRNA on nanoparticle diameter:
  • RNA* RNA*, Biospring
  • siRNA GAA CCA ACU UGC ACA GAG A dTdT(SEQ ID NO: 15); UUG GCG AAG UGA CCA UUA A dTdT(SEQ ID NO : 16)
  • Particle diameter was measured using a Zeta-sizer. Results are mean ⁇ SD from triplicate determinations.
  • Fig. 16 PKDl silencing in Al 72 cells with electroporated protamine/siRNA nanoparticles
  • siRNA concentration 9 ⁇ g/ml
  • C control cells
  • Fig. 17 PKDl silencing in U87MG cells uing a lentiviral system expressiong 5 different shRNAs against PKD 1
  • Fig. 18 Sequence listing SEQ ID NO: 1,2,4,5 and sequences of Homo sapiens protein kinase Dl (PKDl) (SEQ ID NO: 3), Homo sapiens protein kinase Dl (PKDl) mRNA (NM 002742; SEQ ID NO: 6) and reverse complement sequence of Homo sapiens protein kinase Dl (PKDl) mRNA (SEQ ID NO: 7).
  • hybridize refers to the hybridization of a first to a second polynucleotide.
  • hybridization assays and conditions are known to those skilled in the art and can be found, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y., 6.3.1-6.3.6, 1991.
  • Moderate hybridization conditions are defined as equivalent to hybridization in 2X sodium chloride/sodium citrate (SSC) at 3OC, followed by a wash in 1 X SSC, 0.1% SDS at 50 0 C.
  • RNA For example under the aforementioned conditions 10 ⁇ g RNA might be separated by 1 % agarose gel electrophoresis and transferred to a nylon membrane. For detection a [32P]dCTP-labeled probe might be used. Band intensities might be determined using a Storm phosphoimaging system. GAPDH might be used as internal control.
  • Highly stringent conditions are defined as equivalent to hybridization in 6X sodium chloride/sodium citrate (SSC) at 45°C, followed by a wash in 0.2 X SSC, 0.1 % SDS at 65°C.
  • SSC sodium chloride/sodium citrate
  • RNA interference refers to an RNA induced block of gene expression in a specific and post-transcriptional manner by degradation of a specific target mRNA. This process can involve the action of the RNA- induced silencing complex (RISC).
  • RISC RNA- induced silencing complex
  • RNA refers to small interfering RNA and is used herein according to its conventional and well known meaning in the art.
  • the determination of percent identity between two sequences is preferably accomplished using the mathematical algorithm of Karlin and Altschul (1993) Proc. Natl. Acad. Sci USA 90: 5873-5877. Such an algorithm is incorporated into the BLASTn and BLASTp programs of Altschul et al. (1990) J. MoI. Biol. 215: 403-410 available at NCBI (http://www.ncbi.nlm.nih.gov/blast/Blast.cge). The determination of percent identity is performed with the standard parameters of the BLASTn and BLASTp programs.
  • BLAST polynucleotide searches are performed with the BLASTn program.
  • the "Max Target Sequences” box may be set to 100
  • the "Short queries” box may be ticked
  • the "Expect threshold” box may be set to 10
  • the "Word Size” box may be set to 28.
  • the "Match/mismatch Scores” may be set to 1,-2 and the "Gap Costs” box may be set to linear.
  • the "Low complexity regions” box may not be ticked, the "Species-specific repeats” box may not be ticked, the "Mask for lookup table only” box may be ticked, the "Mask lower case letters” box may not be ticked.
  • the "Max Target Sequences” box may be set to 100
  • the "Short queries” box may be ticked
  • the "Expect threshold” box may be set to 10
  • the "Word Size” box may be set to "3”.
  • the scoring parameters the "Matrix” box may be set to "BLOSUM62”
  • the "Gap Costs” Box may be set to "Existence: 11 Extension: 1”
  • the “Compositional adjustments” box may be set to "Conditional compositional score matrix adjustment”.
  • the "Low complexity regions” box may not be ticked
  • the "Mask for lookup table only” box may not be ticked and the "Mask lower case letters” box may not be ticked.
  • polypeptide refers to a polymer of amino acids and does in general not refer to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide.
  • Polypeptides according to the definition may be post-translationally modified for example by glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogues of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
  • subject in the context of the present invention preferably refers to a human. However, veterinary applications are also in the scope of the present invention. The term “subject” can therefore also refer to an animal, preferably a mammal.
  • cancer or tumor as used herein are considered to include different types of cancers such as colorectal, lung and breast cancer and cancers of the brain, such as glioblastoma.
  • Other cancers or tumors may include non-Hodgkin lymphoma, head and neck cancer, non-small cell lung cancer, ovarian cancer or urinary bladder cancer.
  • expression vector refers to a vector that contains a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence and is capable of inducing protein expression in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems.
  • Expression vectors may comprise functional elements such as e.g., a promoter that is operatively linked to the nucleic acid sequence to be transcribed, a termination sequence that allows proper termination of transcription and a selectable marker.
  • a promoter that is operatively linked to the nucleic acid sequence to be transcribed
  • a termination sequence that allows proper termination of transcription and a selectable marker.
  • the expression vector may further comprise an origin of replication (ORI).
  • ORI origin of replication
  • Suitable expression vectors are known to the person skilled in the art. Depending on whether expression is to be achieved in a prokaryotic or eukaryotic host cell or in in vitro expression systems, the vectors may be prokaryotic and/or eukaryotic expression vectors such as plasmids, cosmids, minichromosomes, bacterial phages, retroviral vectors etc. The skilled person will be familiar with how to select an appropriate vector according to the specific need.
  • PKC ⁇ Protein kinase D 1
  • PKC ⁇ Protein kinase D 1
  • the inventors of the present invention have surprisingly found that the expression level of protein kinase D 1 (PKDl, also called PRKDl or PKC ⁇ ) in astrocytoma cells correlates with grading and severity of the astrocytoma.
  • PKI protein kinase D 1
  • WHO grade IV astrocytoma cells exhibit higher PKDl expression levels than WHO grade III astrocytoma cells (see figure 1 in the Example section).
  • PKDl therefore constitutes a new target for tumor therapy, in particular brain tumor therapy.
  • the invention refers to a compound suitable for reducing or inhibiting the expression or activity of a polypeptide according to SEQ ID NO: 3 or a homologue thereof.
  • said compound is an isolated polynucleotide, an aptamere, an antibody or an inhibitory peptide.
  • aptamer refers to a polynucleotide that specifically binds to one or more molecular target molecules
  • a preferred target in the context of the present invention is a polypeptide according to SEQ ID NO: 3 or a homologue thereof.
  • the aptamer might also bind to and interfere with the activity of any polypeptide that is involved in the intracellular expression of a polypeptide according to SEQ ID NO: 3 or a homologue thereof, such as for example a polypeptide that is part of the intracellular transcription or translation machinery.
  • target-specific means that the aptamer binds to the target molecule with a much higher degree of affinity than it binds to contaminating materials.
  • the specificity of the binding is preferably defined in terms of the comparative dissociation constants (Kd) of the aptamer for its ligand as compared to the dissociation constant of the aptamer for other materials in the enviromnent or unrelated molecules in general.
  • Kd comparative dissociation constants
  • the Kd for the aptamer with respect to its ligand will be at least about 10-fold less than the Kd for the aptamer with unrelated material or accompanying material in the environment. Even more preferably, the Kd will be at least about 50-fold less, more preferably at least about 100-fold less, and most preferably at least about 200-fold less.
  • An aptamer will preferably be between about 10 to about 300 nucleotides in length. Preferably an aptamer will be between about 30 to about 100 nucleotides in length. Most preferably an aptamer will be between about 10 to 60 nucleotides in length.
  • Aptamers may be prepared by any known method, including synthetic, recombinant, and purification methods, and may be used alone or in combination with other aptamers specific for the same target.
  • the term "aptamer” also includes peptide aptamers.
  • antibody includes both polyclonal and monoclonal antibodies, as well as variants or fragments thereof, such as Fv, Fab and F(ab)2 fragments that are capable of binding a polypeptide according to SEQ ID NO: 3 or a homologue thereof.
  • antibody also includes single-chain antibodies.
  • An antibody according to the invention preferably binds to a polypeptide according to SEQ ID NO: 3 or a homologue thereof.
  • the antibody might also bind to and interfere with the activity of any polypeptide that is involved in the intracellular expression of a polypeptide according to SEQ ID NO: 3 or a homologue thereof, such as for example a polypeptide that is part of the intracellular transcription or translation machinery.
  • An inhibitory peptide according to the invention is a polypeptide that is capable of specifically binding to and reducing or inhibiting the activity of a polypeptide according to SEQ ID NO: 3 or a homologue thereof.
  • the activity of a polypeptide according to SEQ ID NO: 3 can for example be determined by analysing the serine phosphorylation status in the trans-and/or autophosphorylation domain using phosphosite specific antibodies and western blotting.
  • in vitro phosphorylation assays using ( ⁇ 32 P) ATP as e.g. described in Jaggi et al. Cancer Res. 2005 Jan 15;65(2):483-92 can be used.
  • An inhibitory peptide according to the invention may also be a polypeptide that specifically binds to and reduces the activity of any polypeptide that is involved in the intracellular expression of a polypeptide according to SEQ ID NO: 3 or a homologue thereof, such as for example a polypeptide that is part of the intracellular transcription or translation machinery.
  • PKDl protein SEQ ID NO: 3
  • RNA interference or antisense approaches efficiently reduces glioblastoma cell proliferation.
  • isolated polynucleotides comprising or consisting of either SEQ ID NO: 1, 2, 15 or 16, which are capable of hybridizing to human PKDl mRNA, turned out to be particularly efficient for this process.
  • the invention provides an isolated polynucleotide comprising or consisting of either SEQ ID NO: 1, 2, 15 or 16 or a fragment or derivative thereof, wherein said polynucleotide is suitable for reducing or inhibiting the expression of a polypeptide according to SEQ ID NO: 3 or a homologue thereof.
  • the isolated polynucleotide according to the invention is suitable for the induction of RNA interference in a cell, preferably a tumor cell.
  • the isolated polynucleotide according to the invention can be used in an antisense approach to inhibit translation of PKDl mRNA in a cell, preferably a tumor cell.
  • the isolated polynucleotide of the invention preferably has a length of between 10 and 100, between 12 and 80, between 14 and 60, between 16 and 50, between 17 and 40, more preferably between 18 and 30 nucleotides and most preferably between 18 and 22 nucleotides.
  • the isolated polynucleotide of the invention typically has a length of about 10 to about 500 nucleotides, of about 11 to about 200 nucleotides, of about 12 to about 100 nucleotides, about 13 to about 75 nucleotides or of about 14 to about 50 nucleotides, of about 15 to about 40 nucleotides, of about 16 to about 30 nucleotides or of about 17 to about 25 nucleotides.
  • isolated polynucleotides according to the invention are also applicable in any other antisense technique suitable for reducing or inhibiting gene-expression known to the skilled person.
  • isolated in the context of the present invention indicates that a polynucleotide has been removed from its natural environment and/or is presented in a form in which it is not found in nature.
  • fragment generally refers to a polynucleotide of between 10 and 20 nucleotides in length.
  • a fragment may be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length.
  • a fragment is typically a portion of the polynucleotide it refers to.
  • derivative refers to a polynucleotide sequences that may differ from the polynucleotide sequence it refers to in that one or more nucleotides of the original sequence are substituted by other nucleotides and/or (chemically) modified by methods known to the skilled person, provided that the polynucleotide is still capable of fulfilling its respective function.
  • derivative also includes polynucleotides having linkages between nucleotides that differ from typical linkages.
  • polynucleotides specifically include 2 '-O-methyl-ribo nucleotide, a polynucleotide derivative in which a phosphodiester bond in a polynucleotide is converted to a phosphorothioate bond, a polynucleotides derivative in which a phosphodiester bond in a polynucleotide is converted to a N3'-P5' phosphoroamidate bond, a polynucleotide derivative in which a ribose and a phosphodiester bond are converted to a pep-tide- nucleic acid bond, a polynucleotide derivative in which uracil is substituted with C-5 propynyl uracil, a polynucleotide derivative in which uracil is substituted with C-5 thiazole
  • derivative may further refer to polynucleotides according to the invention which are covalently linked to or admixed with one or more lipid moieties.
  • a “derivative” may for example be a lipid modified siRNA according to the invention.
  • lipid moieties which may be for example cholesterol, lithocholic acid or lauric acid, are covalently linked to the 3' or 5 '-end of the siRNA, most preferably to the 5 '-end.
  • a “derivative” may show at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, even more preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% or at least 98% and most preferably at least 99% sequence identity to the polynucleotide sequence it refers to.
  • reducing or inhibiting refers to a reduction in expression or activity of a polypeptide in a cell preferably by more than 30%, more preferably by more than 40%, more preferably by more than 50%, more preferably by more than 55%, more preferably by more than 60%, more preferably by more than 65%, more preferably by more than 70%, more preferably by more than 75%, more preferably by more than 80% , more preferably by more than 85%, more preferably by more than 90%, even more preferably by more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, and most preferably by more than 98% in comparison to the expression or activity of said polypeptide in a control cell.
  • a control cell can e.g. be a cell that has not been treated with any of the inventive compounds. Comparison is preferably performed under similar experimental conditions.
  • homologue refers to the level of sequence identity between two proteins or polypeptides. Whether or not two proteins or polypeptides are “homologues” is determined by direct comparison of their amino acid sequences. When the sequences of two proteins or polypeptides are directly compared with each other, these proteins or polypeptides are "homologues” if they are at least 40%, preferably at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and most preferably at least 99% identical with each other. Preferably, said level of sequence identity is determined as described above using the BLASTp programme.
  • a "homologue” may for example be a protein or polypeptide in which amino acids have been deleted (e.g., a truncated version of the protein or polypeptide), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl inositol) in comparison to the polypeptide according to SEQ ID NO: 3.
  • a homologue of a polypeptide according to SEQ ID NO: 3 has preferably the same biological function as the polypeptide according to SEQ ID NO: 3.
  • a homologue does not comprise more than 10, 20, 30, 40, 50 or 100 deleted, inserted, inverted, substituted and/or derivatized amino acids.
  • a homologue preferably has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the biological activity in vitro and/or in vivo as the polypeptide according to SEQ ID NO: 3.
  • an isolated polynucleotide according to the invention is a double stranded RNA or a double stranded DNA molecule.
  • the isolated polynucleotide according to the invention is a double stranded siRNA molecule.
  • an isolated polynucleotide according to the invention is a shRNAs, miRNAs, esiRNA or a dicer-substrate 27-mer duplex.
  • a double stranded DNA molecule according to the invention can for example be used for the expression of an RNA molecule capable of reducing or inhibiting the expression of a polypeptide according to SEQ ID NO: 3 or a homologue thereof.
  • the double stranded DNA molecule according to the invention is preferably inserted into an expression vector.
  • an isolated polynucleotide according to the invention is a single stranded RNA molecule or a single stranded DNA molecule.
  • the isolated polynucleotide according to the invention may be a single stranded siRNA molecule.
  • the isolated polynucleotide according to the invention preferably consists of SEQ ID NO: 15 or 16.
  • the isolated polynucleotide according to the invention may also be a morpholino molecule.
  • Morpholino molecules may for example be used in cases where increased stability of the antisense molecule against cellular nucleases is particularly desirable.
  • morpholino molecules are used as single-stranded polynucleotides.
  • heteroduplexes of a Morpholino strand and a complementary polynucleotide strand may be used.
  • morpholino antisense technology will know how to synthesize and use suitable morpholino molecules.
  • cells might be scrape-loaded with morpholino oligonucleotides used at concentrations between 1 and 20 ⁇ M.
  • morpholino oligonucleotides used at concentrations between 1 and 20 ⁇ M.
  • ethoxylated polyethyleneimine complexed morpholino oligonucleotides might be used for cellular delivery. In this setting the concentrations might be approx. 1 ⁇ M.
  • RNA interference i.e. the reduction or inhibition of the expression of a target gene is typically only transient when the antisense molecules are directly applied to cells.
  • an expression vector must include a polynucleotide sequence that encodes a siRNA molecule or siRNA-like transcript.
  • a particularly efficient method is to express a single stranded RNA that forms a hairpin with a loop. When expressed in a cell, the hairpin siRNA is processed to form a functional siRNA.
  • hairpin siRNA inserts have the advantage that only a single pair of oligonucleotides are needed.
  • Complementary oligonucleotides may be designed that encode hairpin sequences specific to the mRNA target, a loop sequence seperating the two complementary domains and a polythymidine tract to terminate transcription.
  • the present invention in one aspect therefore refers to an isolated polynucleotide, comprising (a) a first polynucleotide sequence comprising SEQ ID NO: 4 or a fragment or derivative thereof and a second polynucleotide sequence comprising SEQ ID NO: 1 or a fragment or derivative thereof; or
  • SEQ ID NO: 2 or a fragment or derivative thereof.
  • the aforementioned isolated polynucleotide according to (a) or (b) is integrated into an expression vector that directs intracellular synthesis of a siRNA molecules or siRNA-like transcript.
  • polynucleotide sequences comprising SEQ ID NO: 4 or SEQ ID NO: 5 are reverse and complement to the polynucleotide sequences comprising SEQ ID NO: 1 or SEQ ID NO: 2 respectively.
  • the first polynucleotide sequence is located upstream of the second polynucleotide sequence, i.e. SEQ ID NO: 4 or a fragment or derivative thereof is located upstream of SEQ ID NO: 1 or a fragment or derivative thereof or SEQ ID NO: 5 or a fragment or derivative thereof is located upstream of SEQ ID NO: 2 or a fragment or derivative thereof within the same polynucleotide strand.
  • the first and/or second polynucleotide sequence has a length of between 10 and 100, between 12 and 80, between 14 and 60, between 16 and 50, between 17 and 40, between 18 and 30 nucleotides, more preferably between 18 and 22 nucleotides. Most preferably the first and/or second polynucleotide sequence hast a length of 19 nucleotides.
  • upstream and downstream are used herein according to their conventional and well known meaning in the art.
  • a linker polynucleotide is located downstream of the first polynucleotide sequence and upstream of the second polynucleotide sequence, i.e. a linker polynucleotide is located downstream of SEQ ID NO: 4 or a fragment or derivative thereof and upstream of SEQ ID NO: 1 or a fragment or derivative thereof or downstream of SEQ ID NO: 5 or a fragment or derivative thereof and upstream of SEQ ID NO: 2 or a fragment or derivative thereof.
  • linker polynucleotide refers to a polynucleotide sequence that acts as a molecular bridge to operably link two different polynucleotides sequences, wherein one portion of the linker is operably linked to a first polynucleotide sequence, and wherein another portion of the linker is operably linked to a second polynucleotides sequence.
  • the linker polynucleotide is a DNA sequence.
  • the length of the linker polynucleotide can vary.
  • the linker is preferably 5 to 15 nucleotides in length, more preferably the linker is 6 to 12 nucleotides in length and most preferably it is 6 or 9 nucleotides in length.
  • the linker can in general comprise any suitable nucleotide sequence.
  • the linker comprises the sequence 5"-ttcaagaga-3" or 5"-ctcgag-3 ⁇
  • the aforementioned isolated polynucleotide according to (a) or (b) can be single stranded or double stranded. In a preferred embodiment the isolated polynucleotide according to (a) or (b) is double stranded.
  • the invention in a further aspect provides an expression vector comprising the aforementioned isolated polynucleotide.
  • the vector allows for the production of double stranded RNA (dsRNA).
  • the expression vector may be a prokaryotic or eukaryotic expression vector such as a plasmid, a minichromosome, a cosmid, a bacterial phage, a retroviral vector, such as e.g. a lentiviral expression vector, or any other vector known to the skilled person.
  • the skilled person will be familiar with how to select an appropriate vector according to the specific need.
  • pSUPER OligoEngine, Inc., Seattle, Washington, United States of America
  • the vector itself and the mechanism how the dsRNA is produced by using said vector is described in Brummelkamp et al., 2002, Science, Vol. 296, pages 550-553.
  • Another example of such a vector named pSilencer available from Ambion was developed by Sui et al., 2002, Proc. Natl. Acad. Sci. Vol. 99, pages 5515-5520.
  • Further preferred expression vectors are lentiviral expression vectors.
  • An example of a lentiviral expression vector is the pLKO.l puro vector (Stewart,S.A., et al.,
  • RNAi Lentivirus-delivered stable gene silencing by RNAi in primary cells, RNA, 9,493- 501 (2003);vector available from Sigma-Aldrich).
  • the expression vector according to the invention might therefore be pSUPER or a lentiviral expression vector.
  • the present invention in another aspect refers to a host cell comprising an expression vector according to the invention.
  • the host cell may be a prokaryotic or eukaryotic host cell.
  • Typical prokaryotic host cells include bacterial cells such as e.g. Escherichia coli (E. coli) or Klebsiella species.
  • Typical eukaryotic host cells include yeast cells such as Saccharomyces cerevisiae, insect cells such as SF9 cells, plant cells and mammalian cells such as COS, CHO and HeLa cells.
  • the eukaryotic host cells are glioblastoma cells.
  • the antisense molecules according to the present invention need to be delivered into target cells, preferably into tumor cells.
  • Typical hosts include mammalian species, such as e.g. humans, non-human primates, dogs, cats, cattle, horses, sheep, and the like. A preferred host is human.
  • the polynucleotide can be directly injected into the target cell / target tissue.
  • Other methods include fusion of the recipient cell with bacterial protoplasts containing the nucleic acid, the use of compositions like calcium chloride, rubidium chloride, lithium chloride, calcium phosphate, DEAE dextran, cationic lipids or liposomes or methods like receptor-mediated uptake (e.g.
  • apoA-I-containing nanoparticles might be added to target cells/target tissue where they bind to scavenger receptor class B, type I and deliver oligonucleotides via 'selective uptake' which does not involve endocytosis of the remaining particle) biolistic particle bombardment ("gene gun" method; e.g. oligonucleotides might be coated on gold particles and delivered in vitro, in situ, or in vivo via a 100 - 600 psi helium pulse into the target cells/tissues), infection with viral vectors, electroporation (e.g. via nucleofection where 2x10 6 cells might be resuspended in 100 ⁇ l transfection reagent. Then, 3 ⁇ g of suitable oligonucleotides might be electroporated under suitable conditions) and the like. The person skilled in the art will be familiar with these methods.
  • polynucleotide according to the invention may be complexed to high densitiy lipoproteins for delivery into the cell.
  • the antisense molecules or any other compound according to the present invention suitable for reducing or inhibiting the expression or activity of a polypeptide according to SEQ ID NO: 3 are delivered to the cell using carrier particles.
  • the present invention in one aspect therefore refers to a carrier particle comprising at least one compound according to the invention that is suitable for reducing or inhibiting the expression or activity of a polypeptide according to SEQ ID NO: 3 or a homologue thereof and/or an expression vector or host cell according to the invention.
  • said carrier particle is a liposome, a micelle, a dendrimer or a nanoparticle.
  • the average diameter of said carrier particle is between 10 nm to 10 ⁇ m.
  • liposome and “micelle” are used herein according to their conventional and well known meaning in the art.
  • Dendrimers in the context of the present invention may be any dendrimers known to the skilled person that are suitable for delivery of biological or pharmaceutical material or compounds.
  • One example for such dendrimers are polypropylenimine dendrimers.
  • the carrier particle according to the invention is a nanoparticle.
  • (ab) is at least 70% identical over its entire length to SEQ ID NO: 7; wherein said isolated polynucleotide is capable of reducing or inhibiting the expression of a polypeptide according to SEQ ID NO: 3 or a homologue thereof; (b) a double stranded siRNA molecule capable of reducing or inhibiting the expression of a polypeptide according to SEQ ID No: 3 or a homologue thereof;
  • an isolated polynucleotide according to the invention comprising or consisting of either SEQ ID NO: 1, 2, 15 or 16, or a fragment or derivative thereof or an isolated polynucleotide according to the invention comprising
  • the aforementioned isolated polynucleotide according to (a) is a single stranded RNA or a single stranded DNA molecule that is capable of hybridizing to human PKDl mRNA (SEQ ID NO: 6), thereby inducing RNA interference or any other intracellular antisense mechanism that results in reduction or inhibition of the expression of a polypeptide according to SEQ ID NO: 3 or a homologue thereof.
  • the isolated polynucleotide according to (a) is preferably at least 50%, preferably at least 60%, preferably at least 70%, more preferably at least 75%, more preferably at least 80 %, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and most preferably at least 99% identical over its entire length to the reverse complement sequence of human PKDl mRNA (SEQ ID NO: 7).
  • the isolated polynucleotide according to (a) preferably has a length of between 10 and 100, between 12 and 80, between 14 and 60, between 16 and 50, between 17 and 40, more preferably between 18 and 30 nucleotides and most preferably between 18 and 22 nucleotides.
  • the isolated polynucleotide according to (a) typically has a length of about 10 to about 500 nucleotides, of about 11 to about 200 nucleotides, of about 12 to about 100 nucleotides, about 13 to about 75 nucleotides or of about 14 to about 50 nucleotides, of about 15 to about 40 nucleotides, of about 16 to about 30 nucleotides or of about 17 to about 25 nucleotides.
  • the aforementioned double stranded siRNA molecule according to (b) can be of any sequence that allows the siRNA molecule to induce RNA interference resulting in reduction or inhibition of the expression of a polypeptide according to SEQ ID No: 3 or a homologue thereof.
  • the aforementioned double stranded siRNA molecule according to (b) has a length of between 10 and 100, between 12 and 80, between 14 and 60, between 16 and 50, between 17 and 40, more preferably between 18 and 30 nucleotides and most preferably between 18 and 22 nucleotides.
  • Nanoparticles according to the invention include e.g. solid lipid nanoparticles, metallic nanoparticles, semiconductor nanoparticles, polymeric and biopolymeric nanoparticles.
  • the nanoparticle according to the invention may comprise one or more materials selected from the group consisting of silica, gold, heavy metals, iron oxide, polymers, biocompatible and biodegradable polymers such as e.g. poly(D,L-lactide- co-glycolide), poly ( ⁇ -caprolactone) and poly ( ⁇ -amino esters), proteins, nucleic acids, lipids,(proteo) liposomes, (reconstituted) lipoproteins, combinations thereof or any other suitable material known to the skilled person.
  • the person skilled in the art will know how to select an appropriate material for a given application.
  • the nanoparticles may comprise a metallic core or shell to exploit for optical imaging or Magnetic Resonance Imaging in tumor diagnostics, guided hyperthermia therapy and guided radiation therapy.
  • the surface of the nanoparticle may be modified.
  • the surface of the nanoparticle may be modified, e.g. by coating/linking with folate, antibodies, adjuvants, ligands, antigens, proteins, enzymes, pH sensitive agents or any other suitable material or compound known to the skilled person.
  • a carrier particle according to the invention in addition to the at least one molecule selected from the group of (a) to (d) or any other compound according to the invention that is suitable for reducing or inhibiting the expression or activity of a polypeptide according to SEQ ID NO: 3 or a homologue thereof comprises at least one compound selected from the group of protamine, serum albumin and Interleukin 13 or combinations thereof.
  • a nanoparticle according to the invention is assembled from protamine, human serum albumin and at least one of the molecules according to (a)-(d).
  • Such a nanoparticle is referred to as ternary nanoparticle.
  • the mass ratio of the at least one molecule according to (a)-(d), protamine and human serum albumin is 1 :3:5.
  • the mass ratio of the at least one molecule according to (a)-(d), protamine and human serum albumin is 1 :4:5.
  • a nanoparticle according to the invention is assembled from protamine and at least one of the molecules according to (a)-(d) in a mass ratio of 4:1.
  • Such a nanoparticle is referred to as binary nanoparticle.
  • nanoparticles according to the invention may comprise Inter leukin-13.
  • Interleukin-13 receptor is constitutively overexpressed on a variety of tumor cells, including malignant glioma cells.
  • Interleukin-13 may therefore be coupled to nanoparticles according to the invention in order to improve the efficacy of targeting said nanoparticles to tumor cells.
  • Interleukin 13 may in particular be coupled to the inventive nanoparticles for improving the efficacy of targeting said nanoparticles to malignant glioma cells.
  • the average diameter of ternary nanoparticle is between 80-500 nm at pH 6.5.
  • the average diameter of binary nanoparticles is between 50-250 nm, at pH 6.5.
  • the present invention relates to pharmaceutical composition for the treatment of cancer
  • pharmaceutical composition for the treatment of cancer
  • said pharmaceutical composition comprises at least one compound selected from the group of (a) an isolated polynucleotide which
  • an isolated polynucleotide according to the invention comprising or consisting of either SEQ ID NO: 1, 2, 15 or 16, or a fragment or derivative thereof or an isolated polynucleotide according to the invention comprising
  • the aforementioned pharmaceutical composition according to the invention comprises at least one compound selected from the group of (a)-(f). However, the aforementioned pharmaceutical composition according to the invention may also comprise combinations of the compounds according to (a)-(f).
  • the aforementioned isolated polynucleotide according to (a) is a single stranded RNA or a single stranded DNA molecule that is capable of hybridizing to human PKDl mRNA (SEQ ID NO: 6), thereby inducing RNA interference or any other intracellular antisense mechanism that results in reduction or inhibition of the expression of a polypeptide according to SEQ ID NO: 3 or a homologue thereof.
  • the isolated polynucleotide according to (a) is preferably at least 50%, preferably at least 60%, preferably at least 70%, more preferably at least 75%, more preferably at least 80 %, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and most preferably at least 99% identical over its entire length to the reverse complement sequence of human PKDl mRNA (SEQ ID NO: 7).
  • the isolated polynucleotide according to (a) preferably has a length of between 10 and 100, between 12 and 80, between 14 and 60, between 16 and 50, between 17 and 40, more preferably between 18 and 30 nucleotides and most preferably between 18 and 22 nucleotides.
  • the isolated polynucleotide according to (a) typically has a length of about 10 to about 500 nucleotides, of about 11 to about 200 nucleotides, of about 12 to about 100 nucleotides, about 13 to about 75 nucleotides or of about 14 to about 50 nucleotides, of about 15 to about 40 nucleotides, of about 16 to about 30 nucleotides or of about 17 to about 25 nucleotides.
  • the aforementioned double stranded siRNA molecule according to (b) can be of any sequence that allows the siRNA molecule to induce RNA interference resulting in reduction or inhibition of the expression of a polypeptide according to SEQ ID No: 3 or a homologue thereof;
  • the aforementioned double stranded siRNA molecule according to (b) has a length of between 10 and 100, between 12 and 80, between 14 and 60, between 16 and 50, between 17 and 40, more preferably between 18 and 30 nucleotides and most preferably between 18 and 22 nucleotides.
  • a pharmaceutical dosage form according to the invention can be administered orally, for example in the form of pills, tablets, lacquered tablets, sugar-coated tablets, granules, hard and soft gelatin capsules, aqueous, alcoholic or oily solutions, syrups, emulsions or suspensions, or rectally, for example in the form of suppositories.
  • Administration can also be carried out parenterally, for example subcutaneously, intramuscularly or intravenously in the form of solutions for injection or infusion.
  • Other suitable administration forms are, for example, percutaneous or topical administration, for example in the form of ointments, tinctures, sprays or transdermal therapeutic systems, or the inhalative administration in the form of nasal sprays or aerosol mixtures.
  • Carriers for soft gelatin capsules and suppositories are, for example, fats, waxes, semisolid and liquid polyols, natural or hardened oils, etc.
  • Suitable carriers for the preparation of solutions, for example of solutions for injection, or of emulsions or syrups are, for example, water, physiological sodium chloride solution, alcohols such as ethanol, glycerol, polyols, sucrose, invert sugar, glucose, mannitol, vegetable oils, etc.
  • the pharmaceutical preparations can also contain additives, for example fillers, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, dispersants, preservatives, sweeteners, colorants, flavorings, aromatizers, thickeners, diluents, buffer substances, solvents, solubilizers, agents for achieving a depot effect, salts for altering the osmotic pressure, coating agents or antioxidants.
  • additives for example fillers, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, dispersants, preservatives, sweeteners, colorants, flavorings, aromatizers, thickeners, diluents, buffer substances, solvents, solubilizers, agents for achieving a depot effect, salts for altering the osmotic pressure, coating agents or antioxidants.
  • Pharmaceutical dosage forms in accordance with the invention may preferably be implants that constantly release the inhibitory nucleic acid molecules in accordance with the invention.
  • a pharmaceutical dosage form according to the invention may be administred intravenously, intracranially, adsorbed to slow release wafers, incorporated in gels, via microperfusion, or by convection-enhanced delivery.
  • the present invention relates to the use of a pharmaceutical composition according to the invention for the manufacture of a medicament for the treatment of cancer.
  • the present invention relates to the use of a compound according to the invention that is suitable for reducing or inhibiting the expression or activity of a polypeptide according to SEQ ID NO: 3 or a homologue thereof and/or an expression vector, host cell or carrier particle according to the invention for the manufacture of a medicament for the treatment of cancer.
  • Said cancer may be a colorectal, lung or breast cancer or cancer of the brain, such as glioblastoma.
  • Said cancer may further be non-Hodgkin lymphoma, head and neck cancer, non-small cell lung cancer, ovarian cancer or urinary bladder cancer.
  • said cancer is a glioma.
  • the pharmaceutical composition according to the invention further comprises an active compound suitable for the treatment of cancer.
  • the active compound is a chemotherapeutic agent.
  • chemotherapeutic agents are temozolomide, adriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosine arabinoside ("Ara-C”), cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids, e.
  • paclitaxel Texol, BristolMyers Squibb Oncology, Princeton, NJ
  • toxotere methotrexate
  • cisplatin melphalan
  • vinblastine bleomycin
  • etoposide ifosfamide
  • mitomycin C mitoxantrone
  • vincristine vinorelbine
  • carboplatin teniposide, daunomycin, carminomycin, aminopterin, dactinomycin,mitomycins, melphalan andotherrelated nitrogen mustards and hormonal agents that act to regulate or inhibit hormone action on tumors such as tamoxifen and onapristone.
  • the chemotherapeutic agent is temozolomide.
  • a preferred embodiment further relates to a pharmaceutical composition according to the invention, wherein said cancer is a brain tumor.
  • said brain tumor is a glioma or a meningioma.
  • said glioma is a malignant glioma of WHO grade III or IV.
  • Said meningioma can be benign or malignant.
  • said glioma is an astrocytoma.
  • the present invention relates to a transgenic animal containing an expression vector capable of expressing a polypeptide which is identical to or a homologue of the polypeptide according to SEQ ID NO: 3 and wherein the polypeptide has the molecular function of the polypeptide according to SEQ ID NO: 3.
  • transgenic animals can e.g. be used as model organisms for research purposes. For example, it might be used to study the effects of different expression levels of the polypeptide according to SEQ ID NO: 3 or a homologue thereof on tumor progression in vivo or to identify a modulator of tumor progression in vivo.
  • transgenic animals typically preferred species for such transgenic animals are rodents such as mice and rats.
  • the expression vector may be integrated into the genome of the transgenic animal. Appropriate expression vectors are well known to those of skill in the art.
  • transgenic animals according to the invention may contain selected systems that allow for regulated expression of the polypeptide according to SEQ ID NO: 3 or a homologue thereof.
  • a system is the cre/loxP recombinase system of bacteriophage Pl.
  • cre/loxP recombinase system of bacteriophage Pl.
  • a recombinase system is the FLP recombinase system of S. cerevisiae (O'Gorman et al. Science 251 :1351-1355 (1991).
  • Transgenic animal are preferably rodents with mice and rats being partcilularly preferred.
  • the present invention relates to a method of detecting the presence of a tumor in a biological sample from a subject comprising at least the steps of:
  • NO: 3 or the homologue thereof in comparison to the control indicates the presence of a tumor in the subject.
  • the person skilled in the art will know how to determine the level of expression of a polypeptide according to SEQ ID NO: 3 or a homologue thereof in a sample.
  • the level of expression of a polypeptide according to SEQ ID NO: 3 or a homologue thereof can for example be determined by Western Blot using an anti-PKDl antibody and subsequent quantitaion of expression levels using densitometric evaluation of the bands of interest and comparing their intensities to those of a house keeping protein like GAPDH or actin.
  • the level of expression of a polypeptide according to SEQ ID NO: 3 in a tumor cell may be approximately 1.3 to 4 fold higher than in a control cell.
  • Suitable control cells would e.g. be primary brain cells (e.g. astrocytes) isolated from brain samples obtained during surgery performed on epilepsy, bipolar disorder or schizophrenia patients.
  • said tumor is a brain tumor.
  • said brain tumor is a glioma or a meningioma.
  • said glioma is a malignant glioma of WHO grade III or IV.
  • Said meningioma can be benign or malignant.
  • said glioma is an astrocytoma.
  • the relative level of expression of the polypeptide according to SEQ ID NO: 3 or the homologue thereof in the sample in (b) as compared to the control correlates with tumor grading (see figure 1 for reference).
  • the present invention provides a method for identifying a molecule capable of modulating cell proliferation comprising at least the following steps:
  • the cells from the cell line according to (a) are mammalian cells, such as for example CHO cells, COS cells, HeLa cells, fibroblasts or any other suitable mammalian cells known to the skilled person .
  • the tissue in (a) is from a tumor and the cell line in (a) is a tumor cell line.
  • a suitable tumor cell line may be for example a primary tumor cell line or any immortalized cell line known to the skilled person, such as for example HeLa cells or Jurkat cells.
  • the cell line in (a) is a genetically modified cell line overexpressing a polypeptide according to SEQ ID NO: 3 or a homologue thereof.
  • overexpressing means that a genetically modified cell expresses a higher amount of a polypeptide according to SEQ ID NO: 3 or a homologue thereof than a non-modified control cell under similar experimental conditions.
  • a higher level of expression can for example be achieved by expressing one or more exogenous copies of a polypeptide according to SEQ ID NO: 3 or a homologue thereof in a cell using an expression vector.
  • a higher level of expression can also be achieved by expressing a polypeptide according to SEQ ID NO: 3 or a homologue thereof from an expression cassette integrated into the genome of the cell, wherein, preferably, a strong promoter such as for example a viral promoter, e.g. a VSV or SV40 promoter, drives the expression of the polynucleotide according to SEQ ID NO: 3 or a homologue thereof.
  • Test compounds according to (b) may for example be compounds from libraries such as small compound libraries, nucleic acid libraries, antibody libraries or peptide libraries.
  • the activity or the expression level in (c) might e.g. differ from the activity or expression level in a control by approximately l%-80 %.
  • a suitable control would be, e.g., a cell obtained in (a) that has not been contacted with a test compound.
  • said tumor is a brain tumor.
  • said brain tumor is a glioma or a meningioma.
  • said glioma is a malignant glioma of WHO grade III or IV.
  • Said meningioma can be benign or malignant.
  • said glioma is an astrocytoma.
  • the human GBM cell line A 172 (obtained from American Type Culture Collection, Rockville, MD, USA) was maintained in Dulbeccos Modified Eagle Medium (DMEM) "high glucose” supplemented with 10% fetal calf serum, lOOU/ml penicillin and lOO ⁇ g/ml streptomycin. Cells were incubated in a humidified 37°C / 5% CO 2 incubator. Cells were splitted every 4-7 days and used in experiments for no more than 20 additional passages.
  • DMEM Dulbeccos Modified Eagle Medium
  • Non-specific RNA UAA GGC UAU GAA GAG AUA C (SEQ ID NO: 14); at least 4 mismatches to any human, mouse or rat gene; microarray tested
  • PKDl siRNA target sequence: NM_002742 (SEQ ID NO: 6), human PRKCM, Start: 1893: 5' - GAA CCA AC UU GC ACA GAG A dTdT- 3' (SEQ ID NO: 15) and Start 1197: UUG GCG AAG UGA CCA UUA A dTdT) (SEQ ID NO: 16) was transfected into Al 72 cells by electroporation using Nucleofector technology from Amaxa.
  • Cells were cultured in 75 cm 2 culture flasks supplemented with medium. Before transfection, cells were washed twice with Ix phosphate-buffered saline (PBS), detached with trypsin and centrifuged (900 rpm, 4 min). The supernatant was removed and according to the manufacturer's instructions about 2x10 6 cells were resuspended in 100 ⁇ l transfection reagent (mouse astrocyte nucleofector solution from Amaxa) at room temperature. Then, 11 ⁇ l (3 ⁇ g) siRNA or 11 ⁇ l (3 ⁇ g) scramble RNA were added. The samples were mixed by pipetting and then transferred into Amaxa certified cuvettes.
  • PBS Ix phosphate-buffered saline
  • Tumor tissue specimens were obtained from patients during open surgical resection of astrocytic gliomas.
  • the WHO grade was diagnosed according to perioperative diagnosis on cryostat sections by a neuropathologist. After removal the biopsies were transferred immediately to liquid nitrogen (for protein expression patterns experiments and immunohistochemical characterization) or prewarmed DMEM ,,high glucose” supplemented with 100 U/mL penicillin and 100 ⁇ g/mL streptomycin (for isolation and culture) and transported to the laboratory within 20 minutes.
  • the biopsy material was homogenized in liquid nitrogen, resuspended in PJPA-buffer (Tris-HCl, 50 mM; pH 7.4; NP-40, 1%; Na-deoxycholate, 0.25%; NaCI, 150 mM; EDTA, 1 mM; PMSF, 1 mM; aprotinin, leupeptin, pepstatin, 1 ⁇ g/ml each; Na 3 VO 4 , 1 mM; NaF, 1 mM), sonicated, and centrifuged to remove debris and insoluble proteins. The supernatant was removed, the protein content analyzed using the Bradford method and equal amounts of protein was separated on SDS-PAGE gels prior to immunoblotting.
  • Tris-HCl 50 mM; pH 7.4; NP-40, 1%; Na-deoxycholate, 0.25%; NaCI, 150 mM; EDTA, 1 mM; PMSF, 1 mM; aprotinin, leupept
  • the human biopsy samples (after transportation) were washed in DMEM and blood vessels were removed as best as possible. Then the samples were mechanically homogenized by staggered roller blades, the homogenized material was rinsed with PBS again and plated in medium on 25 cm 2 flasks. After two days in culture the non-adherent material was removed and adherent cells were further cultured. When the cells were confluent, they were trypsinized and transferred to new flasks for further amplification.
  • Tumor tissue specimens were obtained from patients during open surgical resection as described above. Thereafter the tissue samples were embedded in Tissue Tec OCT and serial cryosections (5 ⁇ m) in a cryostat (Microm HM 500 OM; Microm, Walldorf, Germany) were made. Cryosections were collected on glass slides and air dried for 2 h at 22°C. Before staining, the samples were thawed, fixed once more in acetone for 5 min. at 22°C, rehydrated in PBS for 5 min. and blocked with protein block for 15 min.
  • Tissue Tec OCT and serial cryosections (5 ⁇ m) in a cryostat (Microm HM 500 OM; Microm, Walldorf, Germany) were made. Cryosections were collected on glass slides and air dried for 2 h at 22°C. Before staining, the samples were thawed, fixed once more in acetone for 5 min. at 22°C, rehydrated in PBS for 5 min. and blocked with protein
  • siRNA (Sigma AG, Steinheim, Germany) stock solution (containing 5,0 mg/ml protamine in H 2 O) were diluted with distilled water to a volume of 500 ⁇ l. Then 67,4 ⁇ l of siRNA
  • the preparations were diluted with the corresponding cell culture medium.
  • siRNA can be complexed to reconstituted high density lipoproteins (rHDL) which are prepared by the sodium cholate dialysis method [Bergt et al. Biochem. J. (2000) 346 (345-354)] and are used as delivery vehicle to induce RNAi.
  • rHDL high density lipoproteins
  • DPPC siRNA apo A-I are used at molar ratios of 90/10/1. Aliquots of DPPC and siRNA are dried under nitrogen, followed by the addition of Na-cholate. Tubes are vortexed on ice until the solution is optically clear. To the clear solution apoA-I in endotoxin- free buffer is added.
  • rHDL are incubated over night at 37 0 C for 16 h.
  • siRNA- containing rHDL are reisolated by density-gradient ultracentrifugation [Sattler, W., Mohr, D. and Stacker, R. (1994) Methods Enzymol. 233, 469-489].
  • PKDl protein content correlates with tumor grading
  • PKDl is expressed in GFAP-positive cells (astrocytes) and in HLA-DR-positive cells
  • PKDl-GFP PKDl-GFP
  • PDGF induces translocation of PKDl-GFP
  • PKDl-GFP construct was overexpressed in A 172 cells, which were then challenged by the addition of PDGF. Also these experiments indicate recruitment of PKDl to the plasma membrane in a time-dependent manner (Fig. 4).
  • Lysophosphatidic acid affects Al 72 cell growth
  • LPA lysophosphatidic acid
  • LPA activates PKDl in Al 72 and primary glioblastoma (GBM) cells
  • LPA was also found to be a potent activator of PKDl .
  • the Western blots shown in Fig. 6 demonstrate that a 30 min incubation of A 172 and primary glioblastoma cells led to dose-dependent autophosphorylation of S916 in PKDl.
  • siRNA constructs (target sequence: NM_002742 (SEQ ID NO:6), human PRKCM, Start: 1893: 5' -GAA CCA AC UU GC ACA GAG A dTdT- 3' (SEQ ID NO: 15) and Start 1197: UUG GCG AAG UGA CCA UUA A dTdT) (SEQ ID NO: 16)) were ordered from three different commercial suppliers (Dharmacon, D; Qiagen, Q;
  • Biospring, B at three different concentrations (1, 3 and 5 ⁇ g).
  • the efficacy of PKDl silencing was analyzed by Western blotting (total PKDl levels; Fig. 7). These results (shown for the 5' -GAA CCA AC UU GC ACA GAG A dTdT- 3' construct) indicate that siRNA obtained from all three commercial suppliers are approximately equally effective in inducing RNAi o f PKD 1.
  • RNAi on glioblastoma cell growth was assessed in proliferation assays (Fig. 8). These results revealed that siRNAs obtained from all three suppliers efficiently silence PKDl up to four days and significantly impair glioblastoma cell growth by 75, 83, and 95 %, respectively (D, Q, B) when used at 3 ⁇ g/ml. PKD2 and PKD3 expression levels are not affected by PKDl knockdown
  • PKDl knockdown by RNA interference was also established in primary glioblastoma cells by RNA interference. Results of these preparatory experiments clearly indicate that PKDl in primary glioblastoma cells is accessible to RNAi and is significantly downregulated over a time period of at least four days (Fig. 10). Scrambled siRNA was without effect on PKDl expression levels (Fig. 9, lower panel).
  • ABI 1700 microarray (29098 human genes spotted) analysis was performed. These experiments were performed with A 172 glioblastoma cells that were treated with two siRNA constructs (target sequence: NM_002742 (SEQ ID NO: 6), human PRKCM, Start: 1893: 5' -GAA CCA AC UU GC ACA GAG A dTdT- 3'(SEQ ID NO: 15) and Start 1197: UUG GCG AAG UGA CCA UUA A dTdT) (SEQ ID NO: 16)) in the presence of PDGF.
  • a family clustering of differentially regulated genes revealed that major gene clusters involved in signaling and regulation of cell proliferation were up or downregulated in PKDl silenced glioblastoma cells. Details regarding the regulation of individual genes in response to PKDl silencing are given in Tables II and III.
  • Wild type, scrambled- and PKDl-siRNA treated cells were cultured until 80 % confluency (on 75 cm 2 flasks) and incubated in the absence or presence of PDGF (20 ng). After an overnight incubation cells were washed and lysed. Aliquots (50 ⁇ g protein) of the three lysates were labeled with Cy-2 (wild- type), Cy-3 (scrambled) and Cy-5 (silenced), respectively, and mixed with 500 ⁇ g of the unlabeled protein populations. These samples were separated in the first dimension on an IPG strip (pH 3-10) and in the second on 12 % gels. Protein spots were visualized on a Typhoon imager, and analyzed using the DeCyder software.
  • siRNA was assembled with nanoparticles of a mass ratio 1 :3:5 almost quantitatively. However, to introduce a weak positive electrical charge via the positively charged protein protamine, a mass ratio of 1 :4:5 was chosen for future experiments.
  • nanoparticles were electroporated into A 172 cells (Fig. 16) and the effects on PKDl silencing were analyzed by Western blotting. It was demonstrated for the first time that nanoparticles can be transfected by electroporation followed by siRNA release as evident by induction of RNAi.
  • RNAi Lentiviral constructs (Sigma) expressing 5 different stem-loop constructs encoding hairpin RNAs directed against the mRNA of human PKDl were used to induce RNAi. The following sequences were used:
  • TRCN0000002124 (Clone ID: NM 002742.x-672slcl: coding sequence) CCGGCCCACGCTCTCTTTGTTCATTCTCGAGAATGAACAAAGAGAGCGTG GGTTTTT (SEQ ID NO: 17)
  • TRCN0000002125 (Clone ID: NM 002742.x-2498slcl: coding sequence) CCGGCTAAGGAACAAGGGCTACAATCTCGAGATTGTAGCCCTTGTTCCTT AGTTTTT (SEQ ID NO: 18)
  • TRCN0000002126 (Clone ID: NM 002742.x-2978slcl: 3'-UTR) CCGGCCATCTCCTATAATCTGTCAACTCGAGTTGACAGATTATAGGAGAT GGTTTTT (SEQ ID NO: 19)
  • TRCN0000002127 (Clone ID: NM 002742.x-1556slcl: coding sequence) CCGGCGGCACTATTGGAGATTGGATCTCGAGATCCAATCTCCAATAGTGC CGTTTTT (SEQ ID NO: 20) TRCN0000002128 (Clone ID: NM 002742.x-2270slcl: coding sequence)
  • PKDl expression was analyzed on niRNA (quantitative real time PCR; Fig. 17A) and protein level (Western blot experiments; Fig. 17B). Results indicate that this experimental approach is useful to silence PKDl expression on mRNA and protein level. Densitometric evaluation of the western blot and the corresponding relative optical density (ROD) of PKDl is shown in the bar graph in B.
  • G protein-coupled receptor family C, group 5
  • NIMA severe in mitosis gene a
  • Table III List of repressed genes in PKDl -silenced A 172 cells (Microarray analysis).
  • TIMP metallopeptidase inhibitor 3 (Sorsby fundus

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Abstract

La présente invention concerne entre autres le rôle de PKD1 dans l'évolution de tumeurs cérébrales malignes. L'invention porte sur des composés aptes à réduire ou à inhiber l'expression de PKD1. Elle concerne en outre l'utilisation desdits composés pour le diagnostic et le traitement du cancer, en particulier du cancer du cerveau.
PCT/EP2009/056972 2008-06-06 2009-06-05 Composés permettant de réduire ou d'inhiber l'expression de pkd1 pour le diagnostic et le traitement des tumeurs du cerveau WO2009147246A1 (fr)

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US9801953B2 (en) 2012-10-15 2017-10-31 Emory University Nanoparticles carrying nucleic acid cassettes for expressing RNA
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Cited By (7)

* Cited by examiner, † Cited by third party
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WO2011119842A1 (fr) * 2010-03-25 2011-09-29 The J. David Gladstone Institutes Compositions et procédés pour traiter des troubles neurologiques
US20130095113A1 (en) * 2010-03-25 2013-04-18 The J. David Gladstone Institutes Compositions and methods for treating neurological disorders
JP2013529181A (ja) * 2010-03-25 2013-07-18 ザ ジェイ． デヴィッド グラッドストーン インスティテューツ 神経障害を治療するための組成物および方法
US9359445B2 (en) 2010-03-25 2016-06-07 The J. David Gladstone Institutes Compositions and methods for treating neurological disorders
US9801953B2 (en) 2012-10-15 2017-10-31 Emory University Nanoparticles carrying nucleic acid cassettes for expressing RNA
US20170369887A1 (en) * 2016-06-08 2017-12-28 Sookmyung Women's University Industry Academic Cooperation Foundation Therapeutic compositions for breast cancer containing protein kinase D1 inhibitor
US10626397B2 (en) * 2016-06-08 2020-04-21 Sookmyung Women's University Industry Academic Cooperation Foundation Therapeutic compositions for breast cancer containing protein kinase D1 inhibitor

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